A novel protein domain induces high affinity selenocysteine insertion sequence binding and elongation factor recruitment.
Selenocysteine (Sec) is incorporated at UGA codons in mRNAs possessing a Sec insertion sequence (SECIS) element in their 3'-untranslated region. At least three additional factors are necessary for Sec incorporation: SECIS-binding protein 2 (SBP2), Sec-tRNA(Sec), and a Sec-specific translation elongation factor (eEFSec). The C-terminal half of SBP2 is sufficient to promote Sec incorporation in vitro, which is carried out by the concerted action of a novel Sec incorporation domain and an L7Ae RNA-binding domain. Using alanine scanning mutagenesis, we show that two distinct regions of the Sec incorporation domain are required for Sec incorporation. Physical separation of the Sec incorporation and RNA-binding domains revealed that they are able to function in trans and established a novel role of the Sec incorporation domain in promoting SECIS and eEFSec binding to the SBP2 RNA-binding domain. We propose a model in which SECIS binding induces a conformational change in SBP2 that recruits eEFSec, which in concert with the Sec incorporation domain gains access to the ribosomal A site.
Donovan
Jesse
J
Department of Molecular Genetics, Microbiology and Immunology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Caban
Kelvin
K
Ranaweera
Ruchira
R
Gonzalez-Flores
Jonathan N
JN
Copeland
Paul R
PR
D020413:3' Untranslated Regions; D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010445:Peptide Elongation Factors / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry; D017279:Selenocysteine / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid
D020413:3' Untranslated Regions; C535118:EEFSEC protein, human; D010445:Peptide Elongation Factors; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C121530:SECISBP2 protein, human; D017279:Selenocysteine; D000409:Alanine